Infection by an enveloped virus begins with recognition by the virus of certain receptors on the host cell's membrane. Enveloped viruses enter the host cell by a common mechanism, i.e., fusion of the viral membrane with the host cell membrane, often mediated by a surface protein or surface proteins on the virion. The membrane fusion allows the viral genome segments to be released as ribonucleoproteins (RNP). For example, influenza virus gains entry into the host cell through membrane fusion mediated by hemagglutinin (HA), a glycoprotein found on the surface of the influenza viruses. HA contains a protein motif known as the “jelly roll motif,” or the “Swiss roll motif,” which is frequently found in a variety of different structures including the coating proteins of most spherical viruses examined thus far by x-ray crystallography. Typically, jelly roll motifs comprise eight antiparallel β-strands, although any even number of β-strands greater than four can form a jelly roll motif. The jelly roll motif is at least one structural aspect that is thought to be important for the activity of HA.
Once HA is synthesized on membrane bound ribosomes, its polypeptide chain is eventually cleaved into two chains of amino acids, known as HA1 and HA2, which can be held together by disulfide bonds. Three HA monomers (each with one HA1 and HA2) can trimerize and be transported to the plasma membrane, where the HA2 tails anchor the monomers to the membrane, with the large part of the monomers protruding outside of the membrane. It is believed that about 20 residues at the N-terminal end of HA2 are associated with the mechanism by which virus particles penetrate a host cell. This portion on HA2 is known as the fusion peptide.
HA functions in at least two known roles during viral infection. First, HA binds to the cell, and second, HA acts as a membrane fusogen. HA protein binds to sialic acid residues of glycosylated receptor molecules on target cell surfaces. Once bound, the virus can then enter the cell through endocytosis. The sialic acid binding site has been shown by X-ray crystallography to be located at the tip of an HA subunit within the jelly roll motif.
HA also functions as a membrane fusogen. Once viruses bind to and then enter the cell through endocytosis, proton pumps in the endocytic vesicles (that now contain bound viruses) produce an accumulation of protons and thus a drop of the pH inside the vesicles. If the pH drops below about 6, HA can function as a membrane fusogen. Specifically, a pH drop can induce a conformational change in HA. At a pH of above about 6, the fusion peptide attached to the N-terminus is about 100 Å away from the receptor binding site. At a pH of lower than about 6, the N-terminus moves about 100 Å toward the region of the receptor binding site. It is believed that this structural change enables a fusion mechanism whereby HA brings viral and cellular membranes close together and thus allows the release of viral nucleotides into the cell. This structural change is irreversible, since the low pH conformation is more thermostable than the high pH form. It is also believed that the energy gain during this conformational shift is used by HA as fusion energy.
Vaccinations, a common form of influenza prevention, allow for the production of antibodies that may bind near the receptor binding site on a target cell, and thereby limit the ability of the virus to enter the cell. The virus, however, can evade such inhibitory mechanisms through mutations in residues that form the binding site. Typically, however, such mutations are only found at the rim of the sialic acid binding pocket. It is believed that drastic mutations in this binding pocket could prevent the virus from binding to the cell surface receptor protein. This property of HA makes the binding site one of the ideal targets for inhibitory actors. Compounds inhibiting the fusion process mediated by a viral protein have been tested against viral infections.
For example, Triperiden was shown to inhibit influenza virus replication at a concentration of 20 μg/ml (Heider et al., The influence of Norakin on the reproduction of influenza A and B viruses. Arch Virol. 1985; 86(3-4):283-90. PMID: 2415085). This compound was later shown to inhibit hemolysis of red blood cells and the sensitivity of HA1 to trypsin after low pH treatment of HA (Ghendon et al., Haemagglutinin of influenza A virus is a target for the antiviral effect of Norakin. J Gen Virol. 1986 June; 67 (Pt 6):1115-22. PMID: 3711865). Reassortment of the drug sensitive strain with a strain that was not sensitive confirmed that the target sensitive to triperiden was HA. Triperiden-resistant mutations were mapped to HA as well (Prosch et al., Mutations in the hemagglutinin gene associated with influenza virus resistance to norakin. Arch Virol. 1988; 102(1-2):125-9. PMID: 3196166). In a more recent report, it was shown that inhibition of influenza virus replication by triperiden may be due to its ability to lower the internal pH in the prelysosomal compartment (Ott S et al., Effect of the virostatic Norakin (triperiden) on influenza virus activities. Antiviral Res. 1994 May; 24(1):37-42. PMID: 7944312).
An HA inhibitor (BMY-27709) was shown to have an EC50 of 6-8 μM against influenza viruses that have HA subtypes H1 and H2, but not H3 (Luo et al., Characterization of a hemagglutinin-specific inhibitor of influenza A virus. Virology. 1996 Dec. 1; 226(1):66-76. PMID: 8941323). The compound was shown to inhibit virus replication at an early stage and the inhibition was reversible Inhibitor-resistant mutations were found in HA, and the compound inhibited hemolysis (Luo et al., Molecular mechanism underlying the action of a novel fusion inhibitor of influenza A virus. J. Virol. 1997 May; 71(5):4062-70. PMID: 9094684).
A podocarpic acid derivative (180299) was found as an inhibitor of HA from a chemical library screening (Staschke et al, Inhibition of influenza virus hemagglutinin-mediated membrane fusion by a compound related to podocarpic acid. Virology. 1998 Sep. 1; 248(2):264-74. PMID: 9721235). The EC50 of 180299 is 0.01 μg/ml against influenza A/Kawasaki/86, but ≧10 μg/ml against other trains of virus. The target of action was also confirmed to be HA by inhibition of cell fusion and positions of inhibitor-resistant mutation analyses.
Stachyflin, having an EC50 in the μM range against H1 and H2 viruses, but not H3 viruses, was also identified from a screening (Yoshimoto et al., Identification of amino acids of influenza virus HA responsible for resistance to a fusion inhibitor, Stachyflin. Microbiol. Immunol. 2000; 44(8):677-85. PMID: 11021398). HA as the target for Stachyflin was confirmed by time of addition, inhibition of hemolysis and reassortment between subtype H1 and H3.
Inhibitors of HA fusion were identified from a structure-aided approach (Bodian et al., Inhibition of the fusion-inducing conformational change of influenza hemagglutinin by benzoquinones and hydroquinones. Biochemistry. 1993 Mar. 30; 32(12):2967-78. PMID: 8457561; Hoffman et al., Structure-based identification of an inducer of the low-pH conformational change in the influenza virus hemagglutinin: irreversible inhibition of infectivity. J. Virol. 1997 November; 71(11):8808-20. PMID: 9343241). The most effective compound identified (S19) was shown to have an EC50 of 0.8 μM against influenza virus X-31, while its activities on other strains were not reported. Interestingly, a moderate inhibitor (C22), unlike the other inhibitors that prevented the conformational change of HA, facilitated the conformational change at fusion pH and its effect was irreversible. C22 destabilized HA and also inhibited hemolysis, fusion, and viral infectivity. The authors concluded that because C22 does not induce the conformational change at neutral pH, it was conceivable that it might facilitate fusion by destabilizing HA as an effector.
Small molecular compounds targeted fusion proteins of other enveloped viruses have also been shown to be effective antivirals. For instance, BMS-433771 has an EC50 of 0.02 μM against respiratory syncytial virus (RSV), a negative strand RNA virus (Clanci et al., Orally active fusion inhibitor of respiratory syncytial virus. Antimicrob Agents Chemother. 2004 February; 48(2):413-22. PMID: 14742189). BMS-433771 was shown to inhibit virus replication only when added early, and the compound could inhibit syncythium formation of cells induced by RSV. Drug-resistant mutations were mapped only in the F1 subunit of the fusion protein, suggesting that BMS-433771 inhibits the fusion step in RSV replication cycle. It was later shown that BMS-433771 binds near the N-terminal heptad repeat domain, destabilizes the trimer-of-hairpins structure required for fusion, and is effective to inhibit virus infection in rodent models (Clanci et al., Antiviral activity and molecular mechanism of an orally active respiratory syncytial virus fusion inhibitor. J Antimicrob Chemother. 2005 March; 55(3):289-92. PMID: 15681582).
T-20 is a peptide inhibitor of gp41-mediated virus entry that has been approved by FDA for the treatment of HIV infection [Manfredi et al., A novel antiretroviral class (fusion inhibitors) in the management of HIV infection. Present features and future perspectives of enfuvirtide (T-20). Curr Med. Chem. 2006; 13(20):2369-84. PMID: 16918361). T-20 associates with the gp41 helix bundle present in the fusion intermediate.
Small molecular inhibitors that interact with the glycoprotein of HIV have also been developed (Jiang et al., N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion. Antimicrob Agents Chemother. 2004 November; 48(11):4349-59. PMID: 15504864; Frey et al., Small molecules that bind the inner core of gp41 and inhibit HIV envelope-mediated fusion. Proc Natl Acad Sci USA. 2006 Sep. 19; 103(38):13938-43. PMID: 16963566). In the first case, two N-substituted pyrroles were identified from a syncythium formation screen. They inhibit HIV-1 fusion and entry by interfering with the gp41 six-helix bundle formation. The mechanism of action of these compounds were confirmed by time-of-addition experiments that use a HIV entry assay based on a luciferase cell line and cell-cell fusion based on dye transfer. The binding site for these compounds was modeled on the surface of the six-helix bundle, suggesting that they may behave against the HIV glycoprotein similarly as BMS-433771 against the RSV glycoprotein. The second group designed a binding assay in which a five-helix bundle of gp41 may associate with a fluorescently labeled sixth helix. By this assay, a class of compounds was found to block the association of the sixth helix at 5 μM. This action was more dramatic than only interfering with the fusion activity of the viral glycoprotein. These compounds were shown to inhibit fusion as well as HIV replication. Nevertheless, the data further support that inhibition of fusion is a realistic mechanism to inhibit virus replication by small molecular antivirals.
Viral infection is a major threat to human health and results in significant morbidity and mortality worldwide. For example, according to World Health Organization estimates, seasonal influenza epidemics influence 5˜15% of the global populations annually and are responsible for more than 3-5 million hospitalizations and about 250,000 to 500,000 deaths per year (www.who.int/mediacentre/factsheets/fs211/en/index.html). There is a need for novel therapies for treating viral infections.
Novel compounds that inhibit the fusion process mediated by a viral protein represent an important new class of antiviral agents. Such compounds and methods of using the compounds for treating viral infections are described in the present invention.